Cinnamyl phenol antimicrobial agents

ABSTRACT

Substances which are subject to microbial spoilage are preserved by addition of a cinnamyl phenol, e.g., 2-cinnamyl-phenol, 4cinnamyl phenol, 2-methoxy-4-cinnamyl-phenol, 2-cinnamyl-5methoxy-quinol, etc.

l1 States atent .lnrd et al.

[ Feb. 11, 1975 CINNAMYL Pl-IENOL ANTIMICROBIAL AGENTS [75] Inventors: Leonard .lurd, Berkeley; A. Douglas King, .lr., Martinez; William L. Stanley, Richmond, all of Calif.

[73] Assignee: The United States of America as represented by the Secretary of Agriculture, Washington, DC.

[22] Filed: Apr. 17, 1973 [2!] Appl. No.: 351,932

Related US. Application Data [62] Division of Ser. No. 74,485, Sept. 22, 1970, Pat. No.

[52] U.S. Cl 252/404, 260/4595 R, 424/346 [51] Int.Cl ..A0ln 9/26, BOlj 1/18 [58] Field of Search 252/404; 424/346;

[56] I References Cited OTHER PUBLICATIONS Hurd et al., Jour. of Am. Chem. Soc, Vol. 59 (1937), pp. 107-109.

Primary E.taminer-Benjamin R. Padgett Assistant Examiner-Irwin Gluck Attorney, Agent, or Firm-M. Howard Silverstein; Max D. Hensley; William Takacs 1 Claim, N0 Drawings The compounds of the invention display activities which are equal or even superior to those of widelyused antimicrobial agents. This is illustrated by the following: In general, the compounds of the invention are superior to such known agents as phenol, resorcinol, o-phenyl-phenol, and the alkali metal sorbates and benzoates. For instance, compounds of the invention, at concentrations of 12 to 25 ppm, were found to inhibit the growth of four bacteria (Bacillus cereus, Sarcina lutea, Staphylococcus aureus, and Streptococcus lactis) whereas to achieve the same effect it required 100 to 200 ppm of o-phenyl-phenol, more than 800 ppm of potassium sorbate, and more than 1000 ppm of sodium benzoate.

Alkyl 4-hydroxybenzoates are well known to exhibit potent microbial activity. In general, the compounds of the invention display a superior activity as compared to these benzoates wherein the alkyl group contains less than seven carbon atoms. For instance, whereas the compounds of the invention at a concentration of 12 to 25 ppm will inhibit the growth of the four bacteria noted above, it requires concentrations of 100 to 400 ppm for the same result to be achieved with the C -C alkyl 4-hydroxybenzoates. Moreover, it may be noted that the compounds of the invention have an advantage in that their water-solubility is higher than that of the higher alkyl (e.g., heptyl) 4hydroxybenzoates, and that the compounds of the invention do not exhibit the strong and unpleasant odor which characterizes the said benzoates. Also, with respect to some microorganisms, the compounds of the invention inhibit growth when applied at levels less than required with the higher alkyl 4-hydroxybenzoates. This is the case, for example, with bacteria such as Acaligenes faecelis and E. coli, yeasts such as Pichia chodati, Hansenula anomala, and Saccharomyces cerevisiae, and molds including Aspergillus flavus, A. niger, Penicillium chrysogenum, Rhizopus senti, Botrytis cinerea, Byssochlamys fulva, and Alternaria sp.

Of the various compounds included within the scope of the invention, the cinnamyl-substituted monophenols display particularly high antibiotic activity against a large variety of different microorganisms, and therefore are preferred with respect to the cinnamyl derivatives of dior tri-phenols. Coming into special consideration is 4-cinnamyl-phenol because it displays antibiotic activity over a wide range of pH. The cinnamyl derivatives of monophenols are also preferred because they are essentially colorless, whereas those derived from di or tri-phenols exhibit various shades varying from red to brown.

As evident from the explanation immediately following Formula 1 above, the invention includes compounds wherein there may be nuclear lower alkyl or lower alkoxy substituents in addition to the hydroxy groups. In general, the compounds are preferred wherein such additional substituents are absent or, if present, are in small number, e.g., a total of one to two lower alkyl and/or lower alkoxy substituents. Taking the foregoing facts into consideration, we prefer to employ the compounds of the sub-generic category represented by the formula CH -Cll 011-0) wherein R is lower alkyl,

R" is lower alkoxy,

n is an integer from 0 to 2,

m is an integer from 0 to 2, and

the sum of n and m is not more than 2.

The invention encompasses not: only the use of any of the above-described agents individually, but also mixtures thereof.

In preserving substances in accordance with the invention, any of the aforesaid agents or mixtures thereof are incorporated with the substance, using an amount of the agent to inhibit microbial growth. Additional conventional treatments such as dehydration, canning, refrigeration, or freezing may be applied to the substance containing the added agent. The incorporation of the agent with the substance may involve a mixing of the substance and the agent-this is especially suitable where the substance is in liquid or particulate form. Where the substance is in the form of pieces of large dimensions the agent may be incorporated therewith by coating it on the surface of the pieces. For such purpose the agent is preferably dispersed in a carrier-a liquid such as water, alcohol, water-alcohol blends, oils, or a finely-divided solid such as salt, starch, talc, or the like.

The invention is of wide versatility and can be applied for the preservation of all kinds of substances which are normally subject to microbial spoilage-Typical examples of such substances are listed below by way of example. Foodstuffs such as fruits, vegetables, juices, milk, eggs, meat, fish, grains, cereal products, cheese, etc. Animal glues and mucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparations of natural gums such as tragacanth, Arabic, acacia, karaya, locust bean, agaragar, pectin, elgin, etc.; fermentation broths, mashes, and residues from fermentation processes; whey; wines and Vinegars; animal feeds and ingredients of animal feeds such as fish meals, blood meals, feather meal, meat scraps, bone meal, tankage, grains, and oil-seed meals; proteins and protein hydrolysates; textile printing pastes; paints containing proteins or other spoilage dispersing agents; solutions of bark extracts or other tanning agents; molasses; by-products or wastes that contain potentially valuable carbohydrate, proteinous or fat ingredients such as stick liquor, corn steep liquor, fruit cannery wastes, citrus peels, cull fruit and vegetables, tops of root vegetables, distillers slops, pulp liquors, wash water from textile de-sizing operations, waste liquors from wool scouring plants, dairy and slaughter house wastes and liquors, etc.

The compounds of the invention may be synthesized by known proceduresfor example, those disclosed by Hurd et al, Jour. Am. Chem. Soc., Vol. 59, pp. 107-109; Barnes et al, Tetrahedron, Vol. 21, pp. 2707-2715; Jurd, Experientia, Vol. 24, pp. 858-860; Jurd, Tetrahedron, Vol. 25, pp. 1407-1416; and Jurd, Tetrahedron Letters No. 33, pp. 2863-2866. Typically, these syntheses involve condensing a selected phenol (phenol itself, a cresol, resorcinol, guaiacol, hydroquinone monomethyl ether, etc.) with cinnamyl bromide or cinnamyl alcohol.

The invention is further demonstrated by the following illustrative examples. For comparative purposes, various compounds, including some of known antimicrobial activity, are included in the reported experiments.

1 CINNAMYL PHENOL ANTIMICROBIAL AGENTS This is a division of our co-pending application, Ser. No. 74,485, filed Sept. 22, 1970, which issued July 10, 1973, as US. Pat. No. 3,745,222.

A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to the preservation of substances which are normally subject to microbial spoilage. The objects of the invention include the provision of novel processes and compositions for accomplishing such preservation. Further objects of the invention will be evident from the following disclosure wherein parts and percentages are by weight unless otherwise specified. The abbreviation ppm used herein refers to parts per million. Temperatures are given indegrees Centigrade. The symbol 4) is used herein to designate the phenyl radical.

In accordance with the invention it has been found that certain agents exhibit unexpected antimicrobial activity and are useful for preserving all kinds of materials which are normally subject to microbial spoilage. The agents in question are certain cinnamylphenols, and their antimicrobial activity is unusual and unexpected because it is not shared by closely-related phenol derivatives.

Generically, the agents in accordance with the invention have the structure where at least one but not more than three of the R's are hydroxy radicals, and the remainder of the R's are each independently selected from the group consisting of lower alkyl, lower alkoxy, and hydrogen.

The compounds of the invention are especially useful because they are active against many microorganisms in the categories of bacteria, yeasts, and molds. In other words, the compounds are not just active against one or a few organisms; rather, they display broadspectrum antimicrobial activity.

Examples of particular compounds within the scope of the invention are given below by way of illustration and not limitation:

r-Cinnamyl-phenol (also knovm as obtusastyrene) CH -CH Cit-Q3 011 -011 cit-(b Z-Cinnamybphenol Z-Cinnamyl-h-methyl- H -CH Cit-Q phenol II C 3 Q cu 2-Ci.nnamy1- i-mc thoxyphenol z-Methyli-cinnamyb phenol 4-Cinnnmy1-resorcino1 Oil Oli

2Cinnamy1-5-met:hoxyquinol 3 4-Cinnamy1-pyrogal 101 Cll -Cll CH-Qi A series of compounds were assayed for effectiveness against bacteria, molds, and yeasts, using the following test procedure.

All compounds were tested at a concentration of 500 ppm. (w/v). Plates were prepared by adding a measured amount of the candidate compound (in an appropriate solvent, i.e., acetone, ethanol, or water) to 10 ml. of sterilized medium, mixing thoroughly, pouring into 60 X 15 mm. Petri dishes, and allowing the gel to set. The plates were then inoculated with the test organisms. In the case of bacteria and yeasts the inoculation was done by Lederbergs replica plating technique, applying nine bacteria or seven yeasts on each plate. In the case of molds, drops of homogenized culture were TABLE II placed on the surface of the plates, applying three to four molds per plate.

The media used were: plate count agar (Difco) for bacteria; potato dextrose agar (Difco) for yeasts and 5 molds.

10 days and evaluated by comparison with the controls.

The results are expressed on the basis:

+ for effective to inhibit growth 1': for not completely effective; faint growth occurs for ineffective; growth occurs 15 The results are summarized in the following tables.

Activity of Various Compounds (at conc. of 500 ppm) Against Yeasts 4 2-Cinnamyl- Z-Cinnamyl- Cinna- M h. Cinnamyl- Cinnamyl- Cinnamyl- 5-methoxy- 5-methoxy myl Resor- Pyrooxyphenol rcsorcinol pyrogallol quinol quinone euge- Phenol cinol gallol quinol nol Zygusuzc/wm yces juponit'us (undirla Iropir'alis Pirliiu t'lmrlnli Hunrmulu unumulu SIICC/IUIO/ll your L't'rei-is'iut' Sure/1armnyrz's lm'llix Tu n/a urililt Huusenulu mvlliqul Candida (ha/mars! .S'ucrh a rum yves msei Zygosaccharomyccs barkeri TAB LE III Activity of Various Compounds (at conc. of 500 ppm) Against Molds 4- 4- 4- Z-Cinnamyl- Z-Cinnamyl- Cinna- Meth. Cinnamyl- Cinnamyl- Cinnamyl- 5-methoxy- 5-methoxymyl' Resor- Pyro oxyphenol resorcinol pyrogallol quinol quinone euge- Phenol cinol gallol quinol nol Aspergillus flavus i Aspargillus niger i Penicillium chrysogcnum I m Rhizupus .rcnli bnlrylis' cim'reu H).\'.\t)L'/Ilu"l \ki fuli'u i Alu'rnarin .rp. I

TABLE IV Minimal Inhibitory Concentrations (in ppm.) of Various Compounds Against Bacteria 4- 2- 2-Cinnamyl- 4-Methyl- 2-Cinnamlyl- (arthol- P I S dium C' l- 4'methox 4-Cinnamyl- 4-met y p cny o assium o gii il i gli e ri l phenol y phenol phenol phenol sorbate benzoate Bacillus versus 25 l2 25 l2 l2 800 l 000 Sarcina lulea 25 25 25 l2 I2 200 do. do. "ulfl'liffi 25 25 25 I2 I 2 200 (it). do.

TABLE IV-Continued Minimal Inhibitory Concentrations (in ppm.) of Various Compounds Against Bacteria 4- 2- Z-Cinnamyl- 4-Methyl- Z-Cinnamyl- Ortho- Cinnamyl Cinnamyl- 4-methoxy- 4-Cinnamyl- 4-methylphenyl- Potassium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Slreptococcux lav/ix 2 5 I2 25 l2 I2 100- do. do.

200 Acaligenes faecalis 5 200 200 200 200 l00- do. do.

I00 200 I Escherichia coli 50- 200 200 200 200 100- do. do.

EXAMPLE 2 ducted with varying amounts of each compound to de- A series of compounds were assayed for effectiveness against bacteria, molds, and yeasts as set forth in Example l, except that in this case the assays were contermine the minimum concentration thereof required to inhibit growth.

The results are set forth in the following tables.

TABLE V Minimal Inhibitory Concentrations (in ppm.) of Various Compounds Against Yeasts 4- 2- 2-Cinnamyl- 4-Methyl- Z-Cinnamyl- O- Cinnamyl- Cinnamyl- 4-methoxy- 4-einnamyl- 4-methylphenyl- Potasium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Zygosaccharamyces japonicus l2 I2 25 l2 I2 I00- 800 l000 200 Candida tropicalis l2 I2 50 25 25 I00 200- 250- 400 500 Pichia c/mdati 25 5O 200 200 200 I00 800 l000 Hansenula anamala 5O 50 I00 I00 50 I00- 800 do.

200 Saccharomyces cerevisiae 2 5 25 25 800 do.

. 200 Tarula utilis 50 50 200 I00 200 100- 800 do.

TABLE VI Minimal Inhibitory Concentrations (in ppm.) of Various Compounds Against Molds 4- 2- Z-Cinnamyl- 4-Methyl- Z-Cinnamylo- Cinnamyl- Cinnamyl- 4-methoxy- 4-cinnamyl- 4-methylphenyl- Potassium Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Aspergillus flavus I00 100 200 200 200 100 800 l000 Aspergillus niger 50 100 200 200 200 I00 do. do. Penicillimn chrysogenum 50 25- 200 50 I00 50 do. do.

0 Rhizopus .renri 6 50 200 50 I00 I00 200 500 Borrylis' cinerea 25- 12- 200 50 25 12- 200 500 50 25 25 Byssuchlamys fulva 25 25 50 25 25 S0 800 l000 Allernaria sp. 25 25 I00 50 I00 50 400 500 TABLE VII Minimal Inhibitory Concentrations (in ppm.) of 4-Cinnamyl-phenol and Several Alkyl 4-Hydroxybenzoates Against Bacteria, Yeasts, and Molds 4-Cinnamyl- Heptyl Amyl 4- n-Butyl n-Propyl Ethyl 4- Methyl 4- phenol 4-hydroxyhydroxy- 4-hydroxy- 4-hydroxyhydroxyhydroxybenzoate benzoate benzoate benzoate benzoate benzoate Bacillus cereux 25 I2 I00 200 400 200 200 Sarr'ina Iuu'u 25 I2 I00 200 400 do. do. SIaPIlY/Ot'mtlls aureus 2 5 I2 I00 200 400 do. do. Slreplococcux lactis 2 5 I2 I00 200 400 do. do.

TABLE VI] Continued Minimal inhibitory Concentrations (in ppm.) of 4-Cinnamyl-phenol and Several Alkyl 4-Hydroxybenzoates Against Bacteria. Yeasts. and Molds 4-Cinnamyl- Heptyl Amyl 4- n-Butyl n-Propyl Ethyl 4- Methyl 4- phenol 4-hydroxyhydroxy- 4-hydroxy- 4-hydroxyhydroxyhydroxy' benzoate benzoate benzoate benzoate bcnzoate bcnzoate Acaligcnes faecalis 50 100 200 200 200 400 do. do. Escherichia cali 50- l 200 do. 200 400 do. do.

Z ygmaccharom y car juplmicus l 2 12-25 50 100 100-200 200 do. Candida tmpicalis l2 l2-25 50 l00 200 200 do. Pic/iiu chudatl' 25 200 100 100 200-400 200 do. Hauxenulu anumala O 200 100 100 200- 400 do. do. Saccliuromyces cerevisiae 2 5 100 50 100 200 do. do. Torula utilis 50 100 100 200 do. do.

Aspergillus flavus 100 200 100 200 200 do. do. Aspergillus niger 50 do. 100 200 200 do. do. Penicillium chrysogenum 50 do. I00 I00 200 do. do. Rhizopus semi 6 25 50 100 200 do. do. Bolrytis cinerea 25-50 50-l00 50 100 100-200 100 100 Byssoclilumys fulvu 25 200 100 I00 200 200 200 Alternaria sp. 25 50-100 50 50 100-200 100 200 EXAMPLE 3 without added 4-cinnamyl-phenol were inoculated with Two compounds (4-cinnamyl-phenol and 4-cinnamyl-resorcinol) were assayed for effectiveness against bacteria, molds, and yeasts, except that in this case the assays were conducted at different concentrations of each compound and at different pHs in order to determine the minimum concentration of each compound required to inhibit growth at the particular pH.

The results are tabulated below. M

TABLE Vlll "cultures of wild yeast or pure wine yeast (S. cerevisiae) and held at room temperature. The inoculated juices were observed at intervals to detect the time at which fermentation (gas production) began. This is a measure of microbial activity-where the organisms multiply freely. the time for initiation of fermentation is short; conversely, where microbial growth is inhibited the time for initiation of fermentation is long or does not Effect of pH on Minimal Inhibitory Concentration (ppm.) of 4-Cinnamyl-phenol and 4-Cinnamyl-resorcinol 4-Cinnamyl-phenol pH7 pH6 pH5 pH4 pH3pH7pH6pH5 pH4 pH3 Bacillus cereus 25 25 25 12.5 200 Bacillus cereus 25 25 25 12.5 200 200 100 6.25 Sarcintt Iutea 25 25 25 l2.5 100 100 25 Staphylococcus aureus 25 25 25 25 100 100 50 25 Streptococcus lactis 25 25 25 25 100 I00 50 25 Aculigenes faecalis 50 25 25 25 200 200 100 25 Escherichia coli 25 50 5O 25 100 200 100 50 Zygosucc/mromyces jztponicus 12.5 l2.5 12.5 12.5 100 50 50 50 Candida tropicalis 25 25 25 12.5 50 50 50 Pic/tin cliodati 50 50 25 12.5 I00 150 50 Hunsenulu (mama/u 50 50 50 25 100 I00 100 75 Sacc/mromyces cercvisiue 25 25 25 12.5 50 50 100 50 Gtltii'iC/tllltt s11. Torn/u ulilis 50 50 37.5 25 75 50 75 50 AS/Il'l'gl/IIIS flmlls 100 I00 100 50 200 200 200 I00 Aspergillus nigt'r 100 lol) 75 37.5 200 200 I00 I00 Penicilliunt cliryiugmumi 50 50 50 12.5 100 I00 50 50 Rltizvpus semi 25 25 25 |2.5 I00 I00 100 50 Botrytis cincreu 25 25 25 12.5 100 I00 75 50 B \xt.t'oclilam v.r fulvu 25 25 25 12.5 100 I00 75 50 Altcrnltriu sp. 50 12.5 75 50 EXAMPLE 4 6 occur at all.

Samples of fruit juice (grape and apple) with and The results are tabulated below.

TABLE lX influence ol' 4-Cinnamyl-phenol on l-crmcutution of Fruit Juice Inoculated with Yeasts Amount of Time for initiation of fermentation. hours Run Substrate lnoculunt inoculum. Zero l2.5 ppm 25 mt 50 ppm 100 ppm 300 ppm.

cells/ml. 4-CP ot' -l-CP ol' 4-CP of 4-CP of 4-(P of 4-CP 1 Grape juice Wild yeast 10 7 n.d. l2 11.1.1. 44 n.d. 2 do. do. l0" l7 l7 30 62 96 n.d.

TABLE IX Continued Influence of 4-Cinnamyl-phenol on Fermentation of Fruit Juice Inoculated with Yeasts Amount of Time for initiation of fermentation, hours Run Substrate lnoculum inoculum, Zero 12.5 ppm 25 ppm 50 ppm 100 ppm 200 ppm cells/ml. 4-CP of 4-CP of 4-CP of 4-CP of 4-CP of 4-CP 3 do Wine yeast 10- 12 22 '45 146 co co 4 do. do. 10 41 75 co co no on Apple juice Wine yeast 46 42 76 I Explanation of symbols:

4-CP designates 4-cinnamyl-phenol n.d. means not determined means that fermentation did not occur; the g rganisms had beendestroyed.

Having thus described the invention, what is claimed to microbial spoilage, and is; b. a minor proportion, sufficient to inhibit microbial 1. A composition of matter comprising growth, of 2-cinnamyl-5-methoxyquinol. a. a major proportion ofa substance normally subject 

1. A COMPOSITION OF MATTER COMPRISING A. A MAJOR PROPORTION OF A SUBSTRATE NORMALLY SUBJECT TO MICROBIAL SPOILAGE, AND B. A MINOR PROPORTION, SUFFICIENT TO INHIBIT MICROBIAL GROWTH, OF 2-CINNAMYL-5-METHOXYQUINOL. 